Summary

β-Catenin has a central role in the early axial patterning of metazoan
embryos. In the sea urchin, β-catenin accumulates in the nuclei of
vegetal blastomeres and controls endomesoderm specification. Here, we use
in-vivo measurements of the half-life of fluorescently tagged β-catenin
in specific blastomeres to demonstrate a gradient in β-catenin stability
along the animal-vegetal axis during early cleavage. This gradient is
dependent on GSK3β-mediated phosphorylation of β-catenin.
Calculations show that the difference in β-catenin half-life at the
animal and vegetal poles of the early embryo is sufficient to produce a
difference of more than 100-fold in levels of the protein in less than 2
hours. We show that dishevelled (Dsh), a key signaling protein, is required
for the stabilization of β-catenin in vegetal cells and provide evidence
that Dsh undergoes a local activation in the vegetal region of the embryo.
Finally, we report that GFP-tagged Dsh is targeted specifically to the vegetal
cortex of the fertilized egg. During cleavage, Dsh-GFP is partitioned
predominantly into vegetal blastomeres. An extensive mutational analysis of
Dsh identifies several regions of the protein that are required for vegetal
cortical targeting, including a phospholipid-binding motif near the
N-terminus.

Introduction

β-Catenin plays multiple, important roles in patterning animal
embryos. This protein is an essential component of the highly conserved,
canonical Wnt signaling pathway, which has been studied intensively in
embryonic and cancer cells (reviewed by
Huelsken and Behrens, 2002;
Moon et al., 2002). According
to the current view of the canonical pathway, Wnt signaling leads to the
stabilization of cytosolic β-catenin. In the absence of a Wnt signal,β
-catenin is phosphorylated at several N-terminal serine and threonine
residues, first by a priming kinase, casein kinase I (CKI), and then by
glycogen synthase kinase-3-β (GSK3β). Phosphorylation by GSK3β
targets β-catenin for ubiquitination and proteasome-mediated degradation.
Phosphorylation of β-catenin occurs in a multiprotein complex that
includes GSK3β, Axin (a scaffolding protein), the tumor suppressor gene
product adenomatous polyposis coli protein (APC), and several other proteins.
Binding of secreted Wnt ligands to frizzled receptors inhibits the degradation
of β-catenin via the activation (possibly by phosphorylation) of
dishevelled (Dsh). Activated Dsh inhibits GSK3β by a mechanism that
remains unclear but that might involve recruitment of GBP/FRAT1 to the
degradation complex (Ferkey and Kimelman,
2002; Hino et al.,
2003). Increased cytoplasmic pools of β-catenin are thought
to lead to accumulation of the protein in the nucleus, where β-catenin
interacts with LEF/TCF transcription factors and activates the transcription
of Wnt-responsive genes. Although it has sometimes been argued that signaling
may not be regulated by total or nuclear levels of β-catenin (e.g.
Chan and Struhl, 2002), a large
body of evidence from many experimental systems has shown a correlation
between levels of nuclear β-catenin and signaling (see discussion in
Guger and Gumbiner, 2000;
Henderson and Fagotto, 2002;
Tolwinski et al., 2003).

In the sea urchin, β-catenin is required for the formation of endoderm
and mesoderm. Overexpression of proteins that interfere with nuclear
localization and/or function of β-catenin, including cadherins,
GSK3β and a dominant negative form of TCF/LEF, lead to the development of
`dauerblastula' embryos, which lack mesenchyme cells and gut
(Emily-Fenouil et al., 1998;
Wikramanayake et al., 1998;
Logan et al., 1999;
Vonica et al., 2000).β
-Catenin appears to have multiple functions in endomesoderm
specification. In the large micromere-primary mesenchyme cell (PMC) lineage,β
-catenin is an essential activator of a network of early zygotic
transcription factors, including Pmar1, Ets1, Alx1 and T-brain, that regulate
the powerful signaling properties and later morphogenesis of these cells
(Kurokawa et al., 1999;
Fuchikami et al., 2002;
Oliveri et al., 2002,
2003;
Sweet et al., 2002;
Ettensohn et al., 2003). In the
macromeres, β-catenin is required for the activation of genes involved in
endoderm and non-skeletogenic mesoderm specification, including those that
render the cells responsive to micromere-derived signals
(McClay et al., 2000;
Davidson et al., 2002).β
-Catenin also plays an indirect role in ectoderm specification, through
its influence on vegetally derived signals that pattern the overlying ectoderm
(Wikramanayake et al.,
1998).

Although the regulation of nuclear localization and function ofβ
-catenin is a critical feature of early metazoan development, the
underlying mechanisms are not well understood. It has been proposed that the
differential nuclear accumulation of β-catenin is a consequence of
regulated proteolytic degradation along the embryo axis. This hypothesis is
based on experiments demonstrating that overexpression of proteins predicted
to interfere with or enhance β-catenin degradation lead to corresponding
changes in nuclear localization and axis specification (see reviews by
Moon and Kimelman, 1998;
Sokol, 1999). Although
overexpression studies point strongly to differential degradation, there has
been no direct demonstration that the stability of β-catenin varies along
an early embryo axis. One study compared the half-life of β-catenin on
the dorsal and vegetal sides of the cleavage-stage Xenopus embryo
using biochemical methods but reported that β-catenin was highly and
equally stable (t1/2=3.6-3.8 hours) on both sides of the
embryo (Guger and Gumbiner,
2000). Mechanisms other than regulated proteolysis have also been
put forward to account for changes in levels of nuclear β-catenin,
including regulated nuclear import/export and interactions with cytoplasmic
and nuclear anchoring proteins (Henderson
and Fagotto, 2002). One recent study, for example, has argued that
Wnt signaling increases levels of nuclear β-catenin not by regulating
GSK3β-mediated degradation, but by triggering the degradation of a
cytoplasmic anchoring protein (Tolwinski
et al., 2003).

In the present study, we have exploited the optical transparency of the sea
urchin embryo to measure the half-life of β-catenin in specific cell
lineages in vivo. We report a gradient in β-catenin stability along the
animal-vegetal (A-V) axis during cleavage and show that degradation ofβ
-catenin in animal blastomeres is dependent on GSK3β-mediated
phosphorylation of the protein. We find that overexpression of a dominant
negative form of Dsh blocks nuclear accumulation of β-catenin in vegetal
cells and suppresses mesoderm and endoderm formation. Finally, we report that
a GFP-tagged form of Dsh shows a striking, specific localization to the
vegetal cortex of the fertilized egg and early vegetal blastomeres, where we
propose it is locally activated and subsequently functions to stabilizeβ
-catenin. Through a detailed mutational analysis of Dsh, we have
identified several regions that are required for vegetal targeting, including
a short phospholipid-binding motif within the DIX domain of the protein.

Materials and methods

Animals

Adult Lytechinus variegatus were obtained from the Duke University
Marine Laboratory (Beaufort, NC) and Carolina Biological Supply (Burlington,
NC). Spawning was induced by intracoelomic injection of 0.5 M KCl. For most
experiments, embryos were cultured at 23°C in temperature-controlled
waterbaths.

Cloning of LvDsh, LvAxin and LvGSK3β

A 600-bp fragment of LvDsh was cloned by RT-PCR from mesenchyme
blastula-stage cDNA using degenerate primers. 5′- and 3′-RACE were
used to isolate clones containing the remainder of the coding sequence and the
5′ and 3′ untranslated regions. A single cDNA clone encoding a
fragment of Axin from Strongylocentrotus purpuratus was identified in
an EST sequencing project (Zhu et al.,
2001). PCR primers designed from this sequence were used to
amplify a fragment of LvAxin from unfertilized egg-stage cDNA. 5′- and
3′-RACE were used to isolate clones containing the remainder of the
sequence. LvGSK3β was cloned by RT-PCR using the published sequence of
GSK3β from Paracentrotus lividus and primer sequences described
by Emily-Fenouil et al. (1998).
Sequences of LvDsh, LvAxin, and LvGSK3β have been deposited in GenBank
(Accession numbers AY624074, AY624075 and AY624076, respectively).

RNA injection and immunostaining

For mRNA injections, cDNAs were subcloned into pCS2+MT or pCS2+GFP vectors.
Injections of in-vitro transcribed, capped mRNAs were carried out as described
(Sweet et al., 2002).
Immunostaining with anti-β-catenin antibody was performed following the
protocol of Logan et al.
(1999), except that embryos
were fixed for 4 hours. Point mutations in LvDsh were introduced using the
Quick-Change Site-Directed Mutagenesis Kit (Stratagene) and deletions were
generated by PCR.

4-D confocal laser scanning microscopy

Four-dimensional microscopy was carried out using a Bio-Rad MRC-600
laser-scanning microscope equipped with 40× and 60× water
immersion objectives. Z-stacks (20-40 images/stack) of 256×256 pixel
images were collected every 2 minutes with a step size of 4 μm, using
Kalman filtering and the Fast1 scan rate. Acquisition of the stacks was
automated using the SOM program (dlapse) accessible through the CoMos software
package (Bio-Rad). A two-dimensional projection (maximum-intensity method) was
generated from each z-stack and transferred to a Macintosh G4 computer. NIH
Image was used to generate time-lapse sequences of the two-dimensional
projections. Representative (non-time-lapse) images of embryos expressing
various GFP-tagged proteins were obtained by confocal microscopy as described
above, except that z-stacks were collected at a slow scan rate with a step
size of 1-2 μm and an image size of 512×512 pixels.

Measurements of protein half-life in vivo

To calculate the half-life of GFP-tagged proteins, fertilized eggs were
injected with mRNA encoding the protein of interest and allowed to develop for
a period of time sufficient for accumulating levels of the GFP-tagged product
detectable by confocal microscopy. At various times, the translational
inhibitor emetine was added to the cultures (final concentration=100 μM)
and the embryos were cultured continuously in the presence of the inhibitor.
After 30 minutes, when emetine was maximally effective, time-lapse sequences
were collected as described above. Each sequence encompassed 45-60 minutes of
real time. To ensure that the intensity of all fluorescence images was below
saturation, the CoMos software was used to examine histograms of pixel
intensities in the 8-bit images and settings on the MRC-600 were adjusted such
that the brightest pixel in the raw images in each sequence had an intensity
value of <256. From the maximal-intensity projections of each z-stack, NIH
Image was used to measure the mean pixel intensity within selected regions of
each frame. Average pixel intensities were measured in specific cell lineages
by hand-selecting the cells using a free-form tool. These values represented
average pixel intensities over the entire cellular region, i.e. including both
nuclear and cytoplasmic pools of protein. Initial average pixel values (30
minutes after addition of emetine) were set to 1 and the natural log (ln) of
average pixel intensity was plotted versus time using Microsoft Excel. These
plots showed a linear decrease in pixel intensity over time, as expected for
an exponential decay (Fig. 2D).
A trend line was added to each curve and used to determine the R2
value and the equation of the line (y=mx +b). The
slope (m) was used in the equation t1/2=0.693/m
to calculate the half-life (t1/2) of the fluorescence in
each cell lineage.

In-vivo measurements of β-catenin-GFP half-life. (A-C) Measurement ofβ
-catenin-GFP half-life at the 16-cell stage. Confocal projections
(vegetal views) of an embryo expressing Xl-wt-β-catenin-GFP. Emetine was
added immediately after the third cleavage division (A), and after 30 minutes
in the presence of the inhibitor the embryo had completed fourth cleavage (B).
Loss of β-catenin-GFP was apparent in animal blastomeres during the
ensuing 30 minutes (arrows, B,C). (D) Representative data from a single
embryo, showing decay of GFP fluorescence in specific cell lineages as
measured by 4-D confocal microscopy. 0 on the x-axis corresponds to
30 minutes after addition of emetine. (E) Control experiment measuring
35S-methionine incorporation after 25 minutes' exposure to varying
concentrations of emetine. 100 μM emetine blocked ∼90% of new protein
synthesis. (F) Control for GFP photobleaching. Fertilized eggs were injected
with mRNA encoding GFP and allowed to develop to the 8-cell stage. Embryos
were treated with 100 μM emetine for 30 minutes and then imaged with 4-D
confocal microscopy under conditions identical to those shown in
Fig. 1. These representative
data from a single embryo show that GFP fluorescence (mean fluorescent pixel
intensity measured over the entire embryo) remained constant over the period
of the experiment. 0 on the x-axis corresponds to 30 minutes after
addition of emetine. (G) Decay in GFP fluorescence is dependent onβ
-catenin phosphorylation. Fertilized eggs were injected with
Xl-pt-β-catenin-GFP mRNA. At the 8-cell stage, embryos were treated with
100 μM emetine for 30 minutes and then imaged with 4-D confocal microscopy
under conditions identical to those shown in
Fig. 1. These representative
data from a single embryo show that GFP fluorescence (mean fluorescent pixel
intensity measured over the entire embryo) remained constant over the period
of the experiment. (H) Summary of Xl-wt-β-catenin-GFP half-life
measurements. Half-life was measured in different cell lineages of cleavage
stage embryos and data were pooled from 8-, 16- and 64-cell stage embryos. On
average, within any individual embryo, the half-life of β-catenin-GFP in
the vegetal-most blastomeres (the micromere territory) was more than 8-fold
greater than in the animal blastomeres (the mesomere territory).

35S-methionine experiments

Cleavage-stage embryos were incubated in emetine for 25 minutes and 20μ
Ci/ml 35S-methionine was then added to the medium for 15
minutes. After TCA precipitation, incorporation of the radioactive label was
measured with a scintillation counter. Levels of incorporation were plotted
relative to sibling embryos not exposed to emetine.

Results

Dynamics of β-catenin turnover in vivo and regulation by
GSK3β

Using the optically transparent embryos of Lytechinus variegatus,
we expressed GFP-tagged Xenopus β-catenin
(Xl-wt-β-catenin-GFP) and followed the dynamics of protein expression
using 4-D confocal laser-scanning microscopy
(Fig. 1). Microinjection of
in-vitro transcribed, capped mRNA encoding Xl-wt-β-catenin-GFP resulted
in detectable protein expression by the 2-cell stage. Initially, the protein
was expressed at similar levels in all blastomeres
(Fig. 1A); in the cytoplasm and
nucleus, and in association with cell membranes. Later in cleavage, levels of
GFP fluorescence (both cytoplasmic and nuclear) in animal blastomeres declined
dramatically (Fig. 1B-E). Over
1-2 cell cycles, nuclear Xl-wt-β-catenin-GFP became restricted to a
region at the vegetal pole of the embryo that included the micromere territory
and the vegetal-most portion of the macromere territory. This restricted
pattern of Xl-wt-β-catenin-GFP nuclear localization closely matched the
normal vegetal nuclear accumulation of β-catenin in L.
variegatus embryos, visualized using an antibody against the endogenous
protein (Logan et al.,
1999).

Four-dimensional confocal analysis of Xl-β-catenin-GFP expression.
(A-E) Frames from a time-lapse sequence following injection of
Xl-wt-β-catenin-GFP mRNA at the 1-cell stage. Times after the start of
recording (hours:minutes) are shown in the bottom left corner of each panel
and cell number is shown in the bottom right corner. GFP-tagged β-catenin
was initially localized in the nuclei, cytoplasm and junctional complexes of
all blastomeres (A). GFP-tagged β-catenin disappeared from the animal
region of the embryo over a period of approximately two cell cycles (B-E).
GFP-tagged β-catenin eventually became restricted to a small territory of
cells surrounding the vegetal pole (asterisk). (F-I) Frames from a time-lapse
sequence following injection of Xl-pt-β-catenin-GFP at the 1-cell stage.
Mutation of residues phosphorylated by GSK3β and a priming kinase at the
N-terminus of β-catenin blocked the disappearance of GFP-tagged protein
from animal blastomeres. The vegetal pole is marked by an asterisk. (J)
Co-injection of mRNAs encoding Xl-wt-β-catenin-GFP and a kinase-dead,
dominant negative form of GSK3β (Xl-dnGSK3β) at the 1-cell stage.
Expression of dnGSK3β stabilized GFP-tagged β-catenin in animal
blastomeres.

The precise stage at which the loss of β-catenin-GFP in animal
blastomeres was first detectable, and the final boundary of the vegetal domain
of nuclear β-catenin-GFP, varied with the amount of mRNA injected. At
relatively low doses, clearing could be detected as early as the 16-cell
stage, and nuclear β-catenin-GFP became restricted to the micromere
territory and small numbers of veg2-derived cells. At higher doses, loss of
fluorescence in the animal blastomeres was delayed until the 64-128-cell
stages and larger numbers of cells of the veg2 territory retained nuclearβ
-catenin. Overexpression of Xl-wt-β-catenin-GFP produced no
apparent phenotype, despite the transient nuclear localization of the protein
in animal blastomeres.

The loss of β-catenin in animal blastomeres was dependent on
GSK3β, a serine-threonine kinase that phosphorylates β-catenin on
several N-terminal residues and targets the protein for ubiquitination and
degradation. We expressed a GFP-tagged form of Xenopus β-catenin
(Xl-pt-β-catenin-GFP) in which four N-terminal serine and threonine
residues were converted to alanines. Three of these residues are
phosphorylated by GSK3β and one by a priming kinase, casein kinase 1
(Yost et al., 1996;
Liu et al., 2002). This
variant of β-catenin shows increased stability in biochemical assays and
in vivo (Yost et al., 1996;
Guger and Gumbiner, 2000).
Overexpression of Xl-pt-β-catenin-GFP resulted in persistent nuclear
localization of the protein in all blastomeres throughout cleavage and
blastula stages (Fig. 1F-I).
Similarly, coexpression of Xl-wt-β-catenin-GFP and a kinase-dead,
dominant negative form of Xenopus GSK3β (Xl-dn-GSK3β)
prevented the loss of β-catenin from animal blastomeres
(Fig. 1J). Overexpression of
Xl-pt-β-catenin-GFP or Xl-dn-GSK3β resulted in a vegetalized
phenotype, as previously reported
(Emily-Fenouil et al., 1998;
Wikramanayake et al., 1998).
These studies indicated that the restriction of nuclear β-catenin-GFP to
the vegetal region is dependent on GSK3β-mediated degradation of the
protein in animal blastomeres.

Measurements of b-catenin half-life in vivo

To measure the half-life of β-catenin in specific cell lineages, we
injected mRNA encoding Xl-wt-β-catenin-GFP into fertilized eggs, allowed
the protein to accumulate to levels detectable by confocal microscopy, then
blocked further protein translation with emetine. Control experiments showed
that 100 μM emetine inhibited >90% of new protein synthesis within 20
minutes (Fig. 2E). Levels of
fluorescence were quantified over the subsequent 45-60 minutes and the rate of
fluorescence decay was used to calculate the half-life of the protein in
different blastomeres (Fig. 2).
Additional control experiments showed that fluorescence decay was dependent on
GSK3β-mediated phosphorylation of β-catenin and that photobleaching
of GFP was negligible over the time course of our experiments
(Fig. 2F-G).

These measurements provided direct evidence of a gradient in β-catenin
stability along the A-V axis during early cleavage. The average half-life ofβ
-catenin-GFP in the mesomere, macromere and micromere territories was
0.24, 0.59 and 1.60 hours, respectively
(Fig. 2H). In our emetine
experiments, we detected differential degradation of β-catenin as early
as the 8-cell stage (approximately 2 hours postfertilization), the first stage
at which cleavage divisions separated animal from vegetal blastomeres. We also
made half-life measurements at the 16- and 64-cell stages and consistently
observed a gradient in β-catenin stability at these stages. We found no
striking increase or decrease in β-catenin stability within any single
cell lineage between the 8- and 64-cell stages, and therefore data from these
various cleavage stages were pooled in the table shown in
Fig. 2H.

Potential regulators of β-catenin degradation: GSK3β and
dishevelled

The differential stability of β-catenin along the A-V axis suggested
that positive or negative regulators of degradation might be localized (or
activated) in the animal or vegetal regions, respectively. Localized
degradation of GSK3β has been proposed as a mechanism for regulating
nuclear accumulation of β-catenin in the early Xenopus embryo
(Dominguez and Green, 2000). We
cloned GSK3β from L. variegatus (GenBank Accession number
AY624076) and expressed a GFP-tagged form, but half-life measurements showed
that the protein was highly and equally stable in all blastomeres
(Fig. 3). Dorsal translocation
of dishevelled (Dsh) following fertilization has also been proposed to play a
role in Xenopus (Miller et al.,
1999). We cloned Dsh from L. variegatus (GenBank
Accession number AY624074) and found that LvDsh mRNA was ubiquitously
expressed in eggs and early embryos (data not shown). We expressed a
GFP-tagged form of LvDsh (Lv-wt-Dsh-GFP) and observed a striking, vegetal
cortical localization (VCL) of the protein
(Fig. 4). At the light
microscope level, Lv-wt-Dsh-GFP accumulated in punctate structures associated
with the cortical cytoplasm in the vegetal region
(Fig. 4F). We also observed
punctate localization to the perinuclear region of all cells, including animal
blastomeres. In some embryos injected with high concentrations of mRNA, VCL
could be detected even before first cleavage
(Fig. 4A). Continuous
observation of such embryos showed that the first and second cleavage planes
bisected the domain of Lv-wt-Dsh-GFP localization. The third cleavage
unambiguously identified this region as the vegetal pole. At the 16-cell
stage, the VCL domain was inherited by the micromeres and, to a lesser extent,
the macromeres. The VCL domain became more difficult to detect at later
stages, but could be identified in some embryos as late as the 32-cell
stage.

GSK3β-GFP is uniformly stable along the animal-vegetal (A-V) axis
during cleavage. (A,B) Two frames from a time-lapse 4-D confocal sequence
following injection of Lv-GSK3β-GFP mRNA at the 1-cell stage. The protein
is found in all blastomeres during early cleavage and does not appear to be
enriched in a specific subcellular compartment. (C) Quantitation of
Lv-GSK3β-GFP turnover following emetine treatment at the 16-cell stage.
The protein is equally and highly stable in mesomere, macromere and micromere
territories.

Vegetal, cortical targeting of GFP-tagged LvDsh. (A) Fertilized egg.
LvDsh-GFP (wild-type) targeted to one pole of the fertilized egg even before
first cleavage (arrow). (B) Two-cell stage. The first cleavage plane bisected
the zone of cortically localized LvDsh-GFP (arrow). (C) 4-cell stage. (D)
8-cell stage. LvDsh-GFP was concentrated in the vegetal cortex of the four
vegetal blastomeres (arrow), which were slightly smaller than the four animal
blastomeres. (E) 16-cell stage. The region of cortically localized LvDsh-GFP
was inherited by the micromeres (arrow) and, to a lesser extent, the overlying
macromeres. (F) High magnification view of the vegetal cortical region of
vegetal blastomeres at the 8-cell stage, showing the punctate nature of GFP
fluorescence (arrow). All panels show different embryos and all show lateral
views except (C), which is viewed along the A-V axis.

Mutational analysis of LvDsh

Dsh is a multi-domain protein that interacts with many partners
(Penton et al., 2002,
Wharton, 2003). We generated
deletions and point mutations in LvDsh to determine which domains were
required for VCL (Figs 5,
6). Although we did not
quantify expression levels of the various protein constructs by
immunoblotting, all were expressed at levels that were readily detectable by
fluorescence microscopy. It was apparent that vegetal targeting did not
correlate with relative levels of expression based on GFP fluorescence, i.e.
of some constructs that yielded very bright fluorescence, some showed
targeting while others did not, and the same was true of constructs that
showed faint fluorescence. In addition, all constructs were injected over at
least a 10-fold range of concentration and we did not detect changes in the
distribution of GFP-tagged proteins over this range.

Analysis of LvDsh domains required for vegetal, cortical localization
(VCL). Mutant constructs and their designations are indicated. `+' indicates
that VCL was evident in essentially all embryos oriented favorably;
`+/–' indicates that some favorably oriented embryos, but not all,
exhibited VCL. In addition, in those embryos in which VCL was apparent, the
crescent of Dsh-GFP was generally less pronounced than observed following
injection of mRNA encoding wild-type LvDsh-GFP. `–' indicates that
essentially no embryos with unambiguous VCL could be identified.

Selected examples of the localization of GFP-tagged Dsh mutants. Deletion
of the C-terminus does not affect VCL (A, arrow) and deletion of the PDZ
domain only partially abrogates targeting (B, arrow). VCL is completely
blocked by deletion of the DIX domain (C) or the region between the PDZ and
DEP domains (D). (E) VCL is also blocked by introducing two point mutations
(K57A/E58A) into the phospholipid-binding motif within the DIX domain. (F)
Co-injection of LvDsh.WT.GFP and the DIX domain of LvDsh shows that the latter
does not exert its dominant negative effect by blocking VCL.

A putative SH3-binding motif (see
Penton et al., 2002) and the
C-terminal region of LvDsh were completely dispensible for VCL
(Fig. 5; LvDsh.ΔPR1.GFP
and LvDsh.ΔC.GFP, respectively). Deletion of the DEP or PDZ domains
[LvDsh.ΔDEP.GFP and LvDsh.ΔPDZ.GFP, respectively; see also
LvDsh.Δ(DEP+C).GFP)] only partially suppressed VCL. Deletion of the
entire N-terminal region (LvDsh.ΔN.GFP), however, or the DIX domain
(LvDshΔDIX.GFP) alone, completely abolished targeting.

Motifs within the DIX domain have been identified that regulate binding to
actin and vesicles in cultured mammalian cells. Point mutations in the
actin-binding motif (K58A) and vesicle (phospholipid)-binding motif
(K68A/E69A) have been shown to abolish the corresponding binding activities
without compromising the structural integrity of the DIX domain
(Capelluto et al., 2002). These
two motifs are well conserved within the DIX domain of LvDsh. A point mutation
in the actin-binding domain of LvDsh (LvDsh.K47A.GFP) that corresponded to the
K58A mutation described by Capelluto et al.
(2002) did not affect VCL, but
the corresponding double mutation in the phospholipid-binding domain
(LvDsh.K57A/E58A.GFP) completely abolished targeting
(Fig. 5;
Fig. 6E). Two other regions
were identified that were required for targeting: a region between the DIX and
PDZ domains that includes multiple phosphorylation sites
(LvDsh.DIXΔPDZ.GFP) and amino acid sequences other than the proline-rich
region that lie between the PDZ and DEP domains (LvDsh.PDZΔDEP.GFP). The
smallest portion of the LvDsh protein sufficient for VCL consisted of
approximately the N-terminal half of the protein
[LvDsh.Δ(DEP+C).GFP].

Overexpression of a dominant negative form of LvDsh

To test whether Dsh function was required for the vegetal stabilization ofβ
-catenin, we overexpressed the DIX domain alone. This region of Dsh is
required for canonical signaling (Kishida
et al., 1999; Rothbacher et
al., 2000, Penton et al.,
2002) and overexpression of the DIX domain phenocopies Dsh null
mutations, indicating that it acts as a dominant negative
(Axelrod et al., 1998). We
found that overexpression of DIX produced an animalized phenotype that was
indistinguishable from the phenotype observed following overexpression of
cadherin (Wikramanayake et al.,
1998; Logan et al.,
1999), i.e. lack of endoderm and reduction or complete absence of
mesoderm (Fig. 7A). LvDsh DIX
also blocked the accumulation of β-catenin in the nuclei of vegetal
blastomeres, as shown by immunostaining with an antibody against endogenous
Lv-β-catenin (Fig. 7D,E).
At these levels of expression, DIX did not block VCL of Lv-wt-Dsh-GFP
(Fig. 6F), indicating that it
acted by a different mechanism. By contrast to the striking phenotype
resulting from DIX overexpression, overexpression of the PDZ domain of LvDsh
had no effect on embryo morphology (Fig.
7B). Axin, another protein that regulates β-catenin
degradation, also contains a DIX domain. To determine whether the effects of
DIX domain overexpression were specific to LvDsh, we cloned LvAxin (GenBank
Accession number AY624075) and overexpressed the LvAxin DIX domain. Injection
of LvAxin DIX mRNA at concentrations equal to or higher than LvDsh DIX mRNA
produced no apparent phenotype (Fig.
7C).

Dsh function is required for endomesoderm specification and for the
accumulation of β-catenin in the nuclei of vegetal blastomeres. (A)
Injection of mRNA (1.9 mg/ml) encoding the DIX domain of LvDsh resulted in
suppression of endoderm and mesoderm formation and a phenotype
indistinguishable from that produced by overexpression of cadherins or
GSK3β (Emily-Fenouil et al.,
1998; Wikramanayake et al.,
1998; Logan et al.,
1999). (B,C) Overexpression of the PDZ domain of LvDsh (2.0 mg/ml
mRNA) or the DIX domain of LvAxin (3.6 mg/ml mRNA) did not produce apparent
phenotypes. Embryos shown in (A-C) are at 20 hours, 20 hours and 18 hours of
development, respectively. (D,E) Overexpression of the DIX domain of LvDsh
blocked the nuclear accumulation of β-catenin in vegetal blastomeres
(arrow, E), as shown by immunostaining using an anti-Lv-β-catenin
antibody.

Discussion

Our in-vivo measurements provide the first direct demonstration of
differential stability of β-catenin along an axis of an early metazoan
embryo. We have found a gradient in β-catenin half-life along the A-V
axis of the sea urchin embryo at early cleavage stages. On average, the
protein is approximately 8 times more stable in the most vegetal cells, the
micromeres, than in animal blastomeres. This difference in protein stability
in the micromere and mesomere territories is sufficient to produce a>
100-fold difference in protein levels in 2 hours, approximately the time
required for the fertilized egg to reach the 8-cell stage. Averaged over all
cell lineages, our estimates of β-catenin half-life in the early sea
urchin embryo are consistent with those previously reported based on bulk
biochemical analyses of whole Xenopus embryos
(Yost et al., 1996) (see also
Guger and Gumbiner, 2000),
whole Drosophila embryos (Pai et
al., 1997) and tissue culture cells
(Byers et al., 1996).

Several observations indicate that the turnover of Xenopus
wild-type β-catenin-GFP mimics that of the endogenous sea urchin protein.
Most significantly, the pattern of nuclear accumulation of
Xl-wt-β-catenin-GFP we observed (Fig.
1) closely matches the pattern of nuclear localization of
endogenous, sea urchin β-catenin detected by immunostaining
(Logan et al., 1999). In
addition, several studies have shown that homologous Wnt pathway proteins from
vertebrates and sea urchins are functionally interchangeable and therefore
likely to be regulated in similar ways. For example, Xenopus and sea
urchin forms of cadherins, GSK3β and LEF/TCF have been tested in
overexpression studies, and in each case the homologs have similar effects
(Emily-Fenouil et al., 1998;
Wikramanayake et al., 1998:
Logan et al., 1999;
Vonica et al., 2000). In
cnidarians, much more distant relatives of vertebrates than sea urchins, the
Xenopus wild-type β-catenin construct used in this study has
been compared to a GFP-tagged form of the endogenous protein and the two have
identical dynamics (Wikramanayake et al.,
2003).

The findings reported here point to differential proteolysis as the
predominant mechanism controlling the polarized nuclear localization ofβ
-catenin during cleavage. Nevertheless, other mechanisms might operate
in parallel. β-Catenin mRNA is uniformly distributed in the egg and early
embryo (Miller and McClay,
1997), and differential localization of maternal β-catenin
mRNA (or localized transcription of the β-catenin gene) is therefore
unlikely to be a contributing factor. We cannot rule out differential
translation as a contributing mechanism, however, as our mRNA constructs might
lack important translational regulatory elements, and the rate of translation
of endogenous β-catenin mRNA in animal and vegetal blastomeres has not
been measured. If differential translation contributes to the polarized
nuclear accumulation of β-catenin, this effect can be overridden by
experimental manipulations that alter post-translational processing of the
protein. Thus, endogenous β-catenin can be driven into the nuclei of more
animal blastomeres by treating embryos with LiCl, an inhibitor of GSK3β
(Logan et al., 1999). Finally,
another plausible mechanism is regulated nuclear import and/or export,
possibly involving interactions with cytoplasmic or nuclear anchoring proteins
(Henderson and Fagotto, 2002;
Tolwinski et al., 2003). Our
observation that wild-type and hyperstable β-catenin-GFP accumulate in
the nuclei of animal blastomeres (the former transiently and the latter
persistently) argues strongly against the possibility that animal blastomeres
lack factors required for nuclear import or retention. A potential limitation
of overexpression studies, however, is that mechanisms that normally regulate
nuclear import/export of β-catenin might be overwhelmed. For example,
artificially high levels of β-catenin might saturate a cytoplasmic
anchoring protein that normally sequesters β-catenin in the cytoplasm of
animal blastomeres. Although the differential translation and cytoplasmic
sequestration models warrant further study, our findings strongly support the
view that differential degradation is a key mechanism regulating the nuclear
accumulation of β-catenin during cleavage.

The methods used in the present study do not allow us to determine
precisely when the polarity in β-catenin degradation first arises. It is
therefore unclear whether differential stability along the A-V axis involves a
local activation of degradation in the animal hemisphere, an inhibition of
degradation in the vegetal region, or a combination of mechanisms. A
difference in β-catenin stability along the A-V axis is clearly
established by the 8-cell stage, when animal and vegetal blastomeres are first
separated from one another by a horizontal cleavage. The finding that
LvDsh-GFP becomes localized at the vegetal pole even prior to first cleavage,
however, suggests that the molecular machinery underlying differential
degradation is polarized maternally or immediately after fertilization. This
observation also indicates that VCL is not dependent on Wnt signals
transmitted between blastomeres, consistent with other evidence that cell-cell
signaling is not required for vegetal nuclear accumulation of β-catenin
in the sea urchin embryo (Logan et al.,
1999).

Our finding that endomesoderm specification and nuclear accumulation ofβ
-catenin are suppressed by overexpression of the DIX domain of Dsh, a
putative dominant negative form of the protein with respect to canonical Wnt
signaling (Axelrod et al.,
1998), provides the strongest evidence to date that Dsh normally
plays an essential role in early deuterostome embryo polarity. It has been
proposed that Dsh plays a role in axis specification in Xenopus,
based on the finding that Dsh protein becomes concentrated on the dorsal side
of the early embryo (Miller et al.,
1999) (see below). There are also differences in levels of Dsh
phosphorylation along the dorsal-ventral axis
(Rothbacher et al., 2000),
although their functional significance (and the role of Dsh phosphorylation in
general) remains unclear (Wharton,
2003). The function of Dsh in early patterning in Xenopus
has been controversial because dominant negative approaches have not yet
revealed a role for the protein in endogenous axis formation. Dominant
negative forms of Dsh that interfere with Wnt-induced secondary axis formation
do not suppress the formation of the endogenous dorsal axis
(Sokol, 1996). Others have
noted possible technical reasons for the failure of these constructs to
suppress normal axis formation, however. Levels of expression of dominant
negative constructs might be insufficient for competing with maternal pools of
protein, particularly if the maternal proteins are already complexed with
other molecules (Miller et al.,
1999; Rothbacher et al.,
2000).

The molecular mechanism of the dominant negative effect of the DIX domain
is unknown. This is largely because the mechanism by which Dsh stabilizesβ
-catenin has not yet been elucidated. It has been proposed that Dsh acts
by binding to Axin, via the DIX domains of the two proteins. This interaction
might prevent Axin multimerization (Hsu et
al., 1999; Kishida et al.,
1999; Sakanaka and Williams,
1999) and/or recruit GBP/FRAT-1, an inhibitor of GSK3β, to
the degradation complex (Li et al.,
1999; Ferkey and Kimelman,
2002; Hino et al.,
2003). Dsh also forms multimers via the DIX domain, and this might
be important for signaling (Kishida et
al., 1999; Rothbacher et al.,
2000). These observations are consistent with a number of
scenarios by which stray DIX domains might disrupt endogenous Dsh-Dsh,
Dsh-Axin and/or Axin-Axin interactions. For example, DIX might compete with
endogenous Dsh for binding to Axin but be unable to recruit GBP/FRAT-1 to the
degradation complex.

Our observations and those of Miller et al.
(Miller et al., 1999) point to
striking similarities in Dsh localization in early sea urchin and
Xenopus embryos. Miller and co-workers reported that in
Xenopus, endogenous Dsh and a GFP-tagged form of the protein
associate with vesicle-like organelles that translocate from the vegetal pole
of the fertilized egg to the future dorsal side during cortical rotation. In
L. variegatus, LvDsh-GFP is associated with granular or vesicular
structures in the vegetal region and this association is dependent on a
vesicle-binding motif within the DIX domain. The major difference appears to
be that Dsh is not redistributed after fertilization in sea urchin eggs, which
lack a cortical rotation. Although the mutational analysis of Miller et al.
(Miller et al., 1999) was not
as detailed as that presented here, both found that association of Dsh-GFP
with vesicle-like structures was dependent on the DIX domain, but not the
C-terminal region of the protein. One difference between the two studies,
however, is that Miller et al. reported that deletion of the DEP or PDZ
domains eliminated association with vesicles, while we found that deletion of
these domains in LvDsh only partially blocked targeting to the vegetal cortex
and association with vesicles in that region. Dsh has been found associated
with punctate cytoplasmic structures in a variety of cell types
(Axelrod et al., 1998;
Itoh et al., 2000), although
it is not known whether these structures are the same as the granular or
vesicular structures observed in eggs and early embryos.

Significantly, in our experiments, we observed no phenotype associated with
overexpression of full length LvDsh, either untagged or GFP-tagged forms, even
at mRNA concentrations sufficiently high to compromise embryo viability.
Others have shown that animal blastomeres can be converted to more vegetal
fates by overexpression of kinase-dead GSK3β
(Emily-Fenouil et al., 1998), a
mechanism that bypasses Dsh. Moreover, in the present study, we showed thatβ
-catenin can be driven into the nuclei of animal blastomeres by
overexpression of kinase-dead GSK3β or mutation of N-terminal
phosphorylation sites in β-catenin. These findings show that disruption
of β-catenin degradation downstream of Dsh leads to nuclear accumulation
of β-catenin in animal blastomeres and changes in cell fate. Therefore,
our finding that overexpression of wild-type LvDsh does not have the same
effect strongly suggests that the protein is not active in animal cells. We
cannot exclude the possibility that, in our experiments, insufficient levels
of LvDsh were expressed in animal blastomeres to produce effects, although
GFP-tagging confirmed that the protein was expressed persistently in all
cells, including animal blastomeres. An alternative hypothesis, and one that
we currently favor, is that Dsh is activated specifically in the vegetal
region (Fig. 8). This local
activation might involve phosphorylation by a vegetally localized activating
kinase or interactions with other vegetal-specific proteins. In light of such
a model, the targeting of LvDsh-GFP to the vegetal cortex might be interpreted
in one of two ways. First, targeting might be an essential step in Dsh
activation. For example, vegetal targeting might bring Dsh into close
association with a localized, activating kinase, such as Par-1
(Sun et al., 2001).
Alternatively, VCL might be a consequence of the vegetal activation of Dsh. In
that event, VCL could serve to concentrate the protein to effective levels in
vegetal blastomeres, or it might play no functional role. Further studies will
be required to identify the mechanisms and function of the vegetal cortical
localization of Dsh and the putative, local activation of this protein in the
vegetal region of the embryo.

Models of Dsh activation and targeting in the vegetal region of the
unfertilized egg or early embryo. We propose that Dsh is activated
specifically in the vegetal region (see Discussion). A putative, vegetally
localized activator is shown in red, egg vesicles in yellow, and Dsh in blue.
Activated Dsh is shown by a blue star-burst symbol. The activator has not been
identified but might be a maternally localized kinase such as Par-1
(Sun et al., 2001).
Alternatively, a repressor of Dsh activation might be localized (or activated)
in the animal region of the embryo. In Model A, local activation of Dsh
triggers targeting to cortical vesicles. This targeting requires a
phospholipid-binding motif in the DIX domain of the protein. Although the
yellow vesicles are shown localized in the vegetal region of the cell, they
might be more widely distributed and Dsh may associate only with those in the
vegetal region. In Model B, unactivated Dsh in the vegetal region of the
embryo associates with vesicles through the DIX domain, and this association
triggers activation of Dsh by the localized activator, which might be
associated with vesicles or the vegetal cortex of the egg.

Acknowledgments

We thank J. Miller and R. Moon for plasmids encoding
Xl-wt-β-catenin-GFP and Xl-pt-β-catenin-GFP, D. Kimelman for plasmid
encoding Xl-dnGSK3β, and D. McClay for antibody against
Lv-β-catenin. C.A.E. was supported by grants from the NSF and NIH and
A.W. was supported by grants from the NSF and the Hawaii Community
Foundation.

Dominguez, I. and Green, J. B. (2000). Dorsal
downregulation of GSK3β by a non-Wnt-like mechanisms is an early
molecular consequence of cortical rotation in early Xenopus embryos.
Development127,861
-868.

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